This invention relates generally to production methods for recombinant viruses, and more specifically, to methods of producing recombinant adeno-associated viruses.
Adeno-associated virus (AAV) is a replication-deficient parvovirus, the genome of which is about 4.6 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5xe2x80x2 and 3xe2x80x2 ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene [B. J. Carter, in xe2x80x9cHandbook of Parvovirusesxe2x80x9d, ed., P. Tijsser, CRC Press, pp.155-168 (1990)]. It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
When this nonpathogenic human virus infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Production of infectious virus and replication of the virus does not occur unless the cell is coinfected with a lytic helper virus, such as adenovirus or herpesvirus. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and helper virus are produced. The infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or xe2x88x92ss DNA genomes following cell lysis.
AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells. Various groups have studied the potential use of AAV in the treatment of disease states. Progress towards establishing AAV as a transducing vector for gene therapy has been slow for a variety of reasons. While the ability of AAV to integrate in quiescent cells is important in terms of long term expression of a potential transducing gene, the tendency of the integrated provirus to preferentially target only specific sites in chromosome 19 reduces its usefulness.
However, an obstacle to the use of AAV for delivery of DNA is lack of highly efficient schemes for encapsidation of recombinant genomes and production of infectious virions. See, R. Kotin, Hum. Gene Ther., 5:793-801 (1994)]. One such method involves transfecting the rAAV genome into host cells followed by co-infection with wild-type AAV and adenovirus. However, this method leads to unacceptably high levels of wild-type AAV. Incubation of cells with rAAV in the absence of contaminating wild-type AAV or helper adenovirus is associated with little recombinant gene expression. In the absence of rep, integration is inefficient and not directed to chromosome 19.
A widely recognized means for manufacturing transducing AAV virions entails co-transfection with two different, yet complementing plasmids. One of these contains the therapeutic or reporter transgene sandwiched between the two cis acting AAV ITRs. The AAV components that are needed for rescue and subsequent packaging of progeny recombinant genomes are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins. Overexpression of Rep proteins have some inhibitory effects on adenovirus and cell growth [J. Li et al, J. Virol., 71:5236-5243 (1997)]. This toxicity has been the major source of difficulty in providing these genes in trans for the construction of a usefull rAAV gene therapy vector.
There remains a need in the art for the methods permitting the efficient production of AAV and recombinant AAV viruses for use as vectors for somatic gene therapy.
The present invention provides methods which permit efficient production of rAAV, which overcome the difficulties faced by the prior art. This method is particularly desirable for production of recombinant AAV vectors useful in gene therapy. The method involves providing a host cell with
(a) a cre transgene, which permits splicing out of the rep and cap gene inhibitory sequences that when removed lead to activation of rep and cap;
(b) the AAV rep and cap genes, 5xe2x80x2 to these genes is a spacer which is flanked by lox sites;
(c) a minigene comprising a therapeutic transgene flanked by AAV inverse terminal repeats (ITRs); and
(d) adenovirus or herpesvirus helper functions.
Thus, in one aspect, the invention provides a method for producing a rAAV which comprises introducing into a host cell a first vector containing the cre transgene under regulatory control of sequences which express the gene product thereof in vitro, a second vector containing a spacer flanked by lox sites, which is 5xe2x80x2 to the rep and cap genes, and a third vector containing a therapeutic transgene flanked by AAV ITRs. These vectors may be plasmids or recombinant viruses. One of the vectors can be a recombinant adenovirus or herpesvirus, which can provide to the host cell the essential viral helper functions to produce a rAAV particle. However, if all the vectors are plasmids, the cell must also be infected with the desired helper virus. The cell is then cultured under conditions permitting production of the cre recombinase. The recombinase causes deletion of the spacer flanked by lox sites upstream of the rep/cap genes. Removal of the spacer allows the rep and cap genes to be expressed, which in turn allows packaging of the therapeutic transgene flanked by AAV ITRs. The RAAV is harvested thereafter.
In another aspect, the invention provides a method wherein a host cell expressing cre recombinase is co-transfected with a vector carrying a spacer flanked by lox sites 5xe2x80x2 to the rep and cap genes, and a vector containing the therapeutic minigene above. With the provision of helper functions by a means described herein, the cell is then cultured under appropriate conditions. When cultured, the cre recombinase causes deletion of the spacer thus activating expression of rep/cap, resulting in the rAAV as described above.
In yet another aspect, the present invention provides rAAV vectors produced by the methods of the invention.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.